The role of ATP and purinoceptor in osteoblast-like cell proliferation induced by mechanical stimulation

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In human osteoblast-like MG-63 cells, increase of [^3H]thymidine incorporation induced by mechanical compression was inhibited by the P2 purinoceptor antagonist suramin, suggesting that mechanical stress-induced DNA synthesis was partially mediated by P2 receptors, however increase of ATP concentration after compression was not detected in supernatant fluid by our assay. Extracellular ATP increased [^3H]thymidine incorporation and cell proliferation, and synergistically enhanced the platelet-derived growth factor (PDGF)- or insulin-like growth factor-1 (IGF-1)-induced [^3H]thymidine incorporation. ATP-induced [^3H]thymidine incorporation was mimicked by the non-hydrolyzable ATPgS, and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. A P2Y receptor agonist UTP and a P2Y receptor antagonist reactive blue 2 did not affect the [^3H]thymidine incorporation, while the P2Y receptor antagonist PPADS inhibited ATP-induced [^3H]thymidine incorp … More oration, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X_4, P2X_5 and P2X_7 but not P2X_1 and P2X_3 receptors. In fura-2-loaded cells, not only ATP but also UTP increased intracellular Ca^<2+> concentration ([Ca^<2+>]i), and inhibitors for several Ca^<2+>-activated protein kinases had no effect on the ATP-induced DNA synthesis, indicating that a [Ca^<2+>]i increase is not indispensable for the ATP-induced DNA synthesis. ATP increased MAP kinase actively in a Ca^<2+>-independent manner, and synergistically enhanced the PDGF- or IGF-1-induced kinase activity. Further, the MAP kinase kinase inhibitor PD98059 abolished the ATP-induced DNA synthesis.We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors, through P2X receptors by activation MAP kinase in a Ca^<2+>-independent manner, and ATP and purinoceptor could play a important role in osteoblast-like cell proliferation induced by mechanical stimulation. Less